Parkinson's disease (PD) is a progressive neurological disorder of late-middle and old age. Although PD is rarely inherited through autosomal or matrilineal maternal pathways, it most often occurs sporadically without identifiable familial patterns (Simon et al., Neurology 54 (2000) 703). Pathologically, PD involves the loss of a number of neural populations, such as the serotonergic neurons in raphe nuclei, the dopaminergic neurons in substantia nigra and the ventral tegmental area, cholinergic neurons of the basal forebrain, and the noradrenergic neurons in the locus ceruleus.
Idiopathic Parkinson's disease (PD) involves a systemic loss of activity of complex I of the mitochondrial electron transport chain, the target enzyme of the parkinsonism-producing neurotoxin, MPTP. This biochemical lesion plays a key pathogenic role. Transfer of PD mitochondrial DNA to a non-PD cell recapitulates this loss of activity and several other pathogenic features of PD, suggesting that this lesion may arise, at least in part, from mitochondrial DNA.
Many human disorders including idiopathic Parkinson's disease (PD) typically arise without clear patterns of inheritance. The lack of recognizable familial associations has led to consideration of non-genetic factors such as toxins or infectious agents as potential causes. In 1989, heteroplasmic mitochondrial DNA (mtDNA) mutations was proposed as a general genetic model that might explain some idiopathic illnesses (Parker et al., Ann. Neurol. 26 (1989) 719; Parker, In: R. M Pascuzzi, Editors, New Developments in Neuromuscular Disease, Indiana University Printing Services, Bloomington, Ind. (1990) 59; Parker and Swerdlow, J. Clin. Ligand Assay Soc. 23 (2000) 141; Parker and Swerdlow, Am. J. Hum. Genet. 66 (1998) 758). This model predicts that a disorder arising through this genetic mechanism should manifest an electron transport chain (ETC) defect of some sort because all mitochondrial gene products are components of the ETC. This model also predicts that the biochemical lesion is likely to be anatomically generalized because of the universal distribution of the mitochondrial genes. Platelet mitochondria in patients with idiopathic PD were studied and a substantial and specific loss of complex I activity was found. A similar lesion in the substantia nigra in idiopathic PD has been reported.
Loss of complex I activity (NADH:ubiquinone oxidoreductase) probably contributes to the cell loss and dysfunction seen in PD as evidenced by the fact that inhibition of this enzyme causes PD in humans and animal. Activity of this critical redox enzyme is depressed in multiple tissues in PD including platelets, muscle, non-nigral brain, as well as substantia nigra, the conventional focus of PD research. A few studies that relied on assay of tissue homogenates rather than purified mitochondria failed to identify this defect indicating the extreme importance of methodological factors (e.g. Nuerta et al., J. Neurol. Sci. 236 (2005) 49; Simon et al., Neurobiol. Aging, 1 (2004) 71).
This loss of complex I activity arises at least in part from mitochondrial DNA (mtDNA) as demonstrated by studies on cytoplasmic hybrids (cybrids). Cybrids are created by the transfer of either control or PD mitochondria (and their mtDNA) into culturable cells depleted of their own endogenous mtDNA. PD cybrids demonstrate loss of complex I activity and a tendency toward apoptotic cell death as well as other features of PD, such as increased free oxygen radical production, altered calcium homeostasis, increased antioxidant enzymes, inclusion bodies containing synuclein, and fully formed Lewy bodies, the histopathological hallmark of PD. These studies strongly suggest that PD mtDNA encodes pathogenic information and raises the possibility of the presence of pathogenic mtDNA mutations in PD.
A number of studies detected homoplasmic and high-frequency heteroplasmic mtDNA mutations in PD, including deletions and single-nucleotide substitutions, but no solid correlations with the PD phenotype have been found (Mellick et al., J. Neural Trans. 111 (2004) 191; Garcia-Lozano et al., Eur. Neurol. 48 (2002) 34). These studies did not detect low frequency heteroplasmic mutations.
Studies relying on direct sequencing of PCR-amplified mtDNA lack the ability to detect low frequency, heteroplasmic mutations. Detection of such low-abundance mtDNA mutations requires sequencing numerous independent clones of the relevant genes. These mutations may be important since the phenotypic effects of mtDNA mutations in other mitochondrial diseases can manifest at low levels of heteroplasmy. Simon et al. recently conducted a study employing a cloning strategy and found a background of mtDNA mutations in PD and aged controls but failed to correlate any specific mutation with the phenotype (Simon et al., Genomics 73 (2001) 113).
Cybrid studies implicated mitochondrial DNA in the pathogenesis of idiopathic PD but numerous studies of mtDNA sequence failed to identify mutations strongly associated with PD. The studies, however, may not have been carried out in enough depth to identify low frequency heteroplasmic mutations.
All seven complex I genes from brain tissue of idiopathic PD and controls have been sequenced by Applicant in previously unattained depth. PD brains did not contain significantly more mutations than control brain. However, the data presented here suggest that a small region of the mitochondrial ND5 gene from codon 120 to codon 150 harbors mutations segregating PD from control. A less important region was identified in ND2.
Applicant demonstrated the predictive potential of the identified correlation by sequencing this region of ND5 from 8 PD and 8 control brain samples. The presence or absence of heteroplasmic mutations correctly classified 15 out of 16 brain samples.
There is a long felt need in the art for methods of diagnosing and treating Parkinson's Disease, and other diseases and disorders, based on gene mutations. The present invention satisfies these needs.